Past work has demonstrated that release of preformed granules from stimulated cultured (RBL-2H3) mast cells is dependent on protein kinase C (PKC) whereas production of cytokines and release of arachidonic acid is regulated by both PKC and MAP (ERK2) kinase. These responses are dependent also on an increase in cytosolic calcium for activation of secretory machinery and translocation of enzymes and vesicles to the plasma membrane (see reports Z01 HL 00990-11 and 00993-11 LMI for details). In the case of antigen-stimulated cells, these processes are mediated through recruitment of the tyrosine kinases, Lyn and in turn Syk, which leads to Syk phosphorylation of phospholipase (PL) PLCgamma and other proteins that initiate the ERK2 cascade, phosphorylation of PLA2 by ERK2, and activation of certain isozymes of PKC by calcium and diacylglycerol (see Beaven and Baumgartner, Curr. Opin. Immunol. 8:766, 1996). Last year, we reported that activation of PKC and secretion is dependent on the activation of a membrane-associated cholera-toxin- sensitive PLD when cells are stimulated via receptors and Ca2+- mobilizing agents. This enzyme, which appeared distinct from the cloned ARF-regulated Golgi-associated PLD (PLD1), can be activated by PKC, elevated free calcium or beta gamma subunits of trimeric G proteins and is regulated by phosphatidylinositol 3-kinase. Ongoing studies have shown that it is not affected by ARF itself or by stimulants (GTPgammaS) and inhibitors (brefeldin A) of ARF and experiments with various combinations of G protein beta gamma subunits (from an insect expression system) are ongoing. Also preliminary cloning experiments indicate expression of PLD1 isoforms in RBL-2H3 cells. As a new initiative, we are cloning the gene for sphingosine kinase as a potential regulator of calcium release in RBL-2H3 cells (Choi et al., Nature 380:634, 1996). A gene that encodes a protein with sphingosine kinase activity and that shows homology to the phosphatidylinositol 3-kinase family of enzymes has been identified. With respect to the MAP kinases, we now find that all currently described mammalian MAP kinase cascades (ERK, p38 MAP kinase and JNK) are activated in antigen-stimulated cells but only the ERK cascade appears to be essential for release of arachidonic acid and the cytokine, TNFalpha (as indicated by selective inhibitors and expression of negative mutants of ERK). The ERK cascade, contrary to published reports, is activated synergistically via calcium and PKC in addition to a third pathway through Shc, Grb2, Sos and Ras. Interestingly, antigen stimulates ERK2 transiently via PKC and then via a long-lived signal, most likely via Shc (see report Z01 HL 0090-11 LMI for further details). The p38 MAP kinase pathway is activated markedly (<10 fold greater than antigen) by stress factors and suppresses activation of ERK. The emerging scenario from our and other studies is that the MAP kinase pathways perform unique and quite distinct functions.